The calyptra influences the plane of division in guard cell mother cells of Funaria and Physcomitrium. Normally, capsules expand while sheathed by the calyptra and the axes of the stomata are parallel to the axis of the capsule in both genera.

Removal of the calyptra from an elongating sporophyte leads to seta thickening prior to capsule expansion and an essentially random orientation of stomata.

If the calyptra is removed from a sporophyte of Funaria at the time the division of the guard cell mother cells is expected, guard cells of abnormal shape and undivided guard cell mother cells are found in unusually high frequency.

Phylogeny of major groups of mosses with the presence of stomata indicated by open circles. Taxa in which the sporophyte is enclosed within the epigonium until after meiosis are underlined. (A) Hypothesis in which there is a single origin of stomata from which pseudostomata of Sphagnum were derived. (B) Hypothesis in which stomata evolved twice and in which pseudostomata are not homologous to stomata.

A moss sporophyte inherits a haploid set of genes from the maternal gametophyte to which it is attached and another haploid set of genes from a paternal gametophyte. Evolutionary conflict is expected between genes of maternal and paternal origin that will be expressed as adaptations of sporophytes to extract additional resources from maternal gametophytes and adaptations of maternal gametophytes to restrain sporophytic demands.

Interpretation

The seta and stomata of peristomate mosses are interpreted as sporophytic devices for increasing nutrient transfer. The seta connects the foot, where nutrients are absorbed, to the developing capsule, where nutrients are needed for sporogenesis. Its elongation lifts stomata of the apophysis above the boundary layer, into the zone of turbulent air, thereby increasing the transpirational pull that draws nutrients across the haustorial foot. The calyptra is interpreted as a gametophytic device to reduce sporophytic demands. The calyptra fits tightly over the intercalary meristem of the sporophytic apex and prevents lateral expansion of the meristem. While intact, the calyptra delays the onset of transpiration.

Predictions

Nutrient transfer across the foot, stomatal number and stomatal aperture are predicted to be particular arenas of conflict between sporophytes and maternal gametophytes, and between maternal and paternal genomes of sporophytes.

Evolution of stomata in mosses (Bryophyta): From molecules to form and function

Abstract

As one of the first land plant groups to diversify, mosses are central in understanding the origin, diversification, and early function of stomata. Unlike tracheophytes that have stomata on anatomically complex leaves and stems, mosses bear stomata exclusively on spore-bearing organs (capsules). However, stomata do not occur in all mosses and, indeed, are absence in the earliest-divergent mosses (Takakia, Andreaea, Andreaeobryum and Sphagnum), suggesting that stomata originated in mosses independently of other plants. The occurrence of structurally unique pseudostomata in Sphagnum further confounds the resolution of homology of moss stomata with those of other plants. The five studies included in this dissertation are aimed at clarifying the structure, development and evolution of moss stomata. The first study focuses on the sporophyte anatomy and stomatal ultrastructure in two structurally and phylogenetically divergent mosses, Oedipodium and Ephemerum. Oedipodium is the sister to peristomate mosses and the first extant moss with true stomata. This monospecific genus has an elaborated capsule with an extended apophysis bearing numerous long-pored stomata. In contrast, Ephemerum nests within the peristomate mosses and has a reduced capsule that lacks an apophysis and has a few round-pored stomata. Ultrastructure of stomata is similar in these two mosses and comparable to that of tracheophytes, except that the stomata of mosses are not as structurally distinct from epidermal cells as are tracheophyte stomata. Anatomical features such as the presence of a cuticle, water-conducting cells, and spongy tissues with large areas for gas exchange are more pronounced in Oedipodium sporophytes and support the role of stomata in gas exchange and water transport during development and maturation. The second study examines changes in pectin composition during development in the model moss Funaria. Stomatal movement in tracheophytes requires guard cell walls to be strong, yet flexible, because they have to undergo reversible deformation to open and close the pore. Pectins are necessary for wall flexibility and proper stomatal functioning in seed plants. In this study of Funaria, immunogold-labeling using five antibodies to pectin epitopes was conducted on guard cell walls during development to relate these features to the limited movement of stomata in moss. Movement of Funaria stomata coincides with capsule expansion when guard cell walls are thin and pectinaceous. Walls dramatically increase in thickness after pore formation and the pectin content significantly decreases in mature guard cell walls, suggesting that a decrease in flexibility is responsible for the inability to open a close previously reported in older moss guard cells. Because this was the first study to demonstrate changes in pectin composition during stomatal development in any plant, a similar study was done on Arabidopsis to identify the main types of pectins in guard cell walls. Localization of pectins in guard cell walls of Arabidopsis is similar to mosses in the stage they can move, with homogeneous walls rich in arabinan pectins that are required for wall flexibility. This study extends knowledge of pectin composition from stomata of the moss Funaria with limited stomatal movement to an angiosperm in which stomatal activity is crucial to the physiological health of the plant. The fourth study describes stomata development and internal changes in sporophyte anatomy that lead to formation of air spaces in the moss Funaria. Developing sporophytes at different stages were examined using light, fluorescence and electron microscopy; immunogold-labeling was used to investigate the presence of pectin in the newly formed cavities. Stomata in mosses do not develop from a self-generating meristemoid like in Arabidopsis, but instead they originate from a protodermal cell that differentiates directly into a guard mother cell. Epidermal cells develop from protodermal or other epidermal cells, i.e., there are no stomatal lineage ground cells. This developmental pattern is congruent with the presence of a gene ortholog of FAMA, but not SPCH and MUTE, in Physcomitrella. The final study in this dissertation focuses on the enigmatic Sphagnum. Although true stomata are absent in early-divergent mosses, Sphagnum has specialized epidermal cells, pseudostomata, that partially separate but do not open to the inside. To further understand the structure, function and evolution of pseudostomata, capsule anatomy and ultrastructure of pseudostomata were detailed. As in moss stomata, pseudostomata wall architecture and behavior facilitate capsule dehydration, shape change, and dehiscence, supporting this common function. Unlike other moss stomata, pseudostomata collapse along their ventral walls and they lack a substomatal cavity. Similarities to true stomata include two modified epidermal cells with specialized cell walls that separate by cuticle deposition and respond to drying. Pseudostomata may be interpreted as modified stomata that suppressed substomatal cavity formation, which in turn eliminated pore development. However, clarification of the homology of pseudostomata and moss stomata will require genomic studies integrated with physiological and structural data. The studies described in this dissertation significantly advance our understanding of moss stomatal development and structure, and provide a comparison point to better evaluate the evolution of stomata. Moss capsule anatomy coupled with the exclusive existence of stomata on capsules supports the concept that stomata in moss are involve in gas exchange but also facilitate drying and dispersal of spores. Changes in wall architecture coupled with a decrease in total pectin explain the inability of mature stomata to move. Development and distribution of stomata in Funaria provides evidence of a direct and less elaborated mechanism for stomatal development than described in Arabidopsis. Resolving relationships among early land plants, especially hornworts and mosses, the only bryophyte groups with stomata, is critical to understanding stomata evolution. Evaluated together, the results of this dissertation are consistent with a single origin of stomata in land plants.

Stomata are key innovations for the diversification of land plants. They consist of two differentiated epidermal cells or guard cells and a pore between that leads to an internal cavity.

Mosses and hornworts are the earliest among extant land plants to have stomata, but unlike those in all other plants, bryophyte stomata are located exclusively on the sporangium of the sporophyte.

Liverworts are the only group of plants that are entirely devoid of stomata.

Stomata on leaves and stems of tracheophytes are involved in gas exchange and water transport.

The function of stomata in bryophytes is highly debated and differs from that in tracheophytes in that they have been implicated in drying and dehiscence of the sporangium.

Over the past decade, anatomical, physiological, developmental, and molecular studies have provided new insights on the function of stomata in bryophytes.

In this review, we synthesize the contributions of these studies and provide new data on bryophyte stomata. We evaluate the potential role of stomata in moss and hornwort life histories and we identify areas that will provide valuable data in ascertaining the evolutionary history and function of stomata across land plants.

Structure and development of walls in Funaria stomata.

The development and structure of the guard cell walls of Funaria hygrometrica Hedw. (Musci) were studied with the light and electron microscopes.

The stoma consists of only one, binucleate guard cell as the pore wall does not extend to the ends of the cell. The guard cell wall is thinnest in the dorsal wall near the outer wall but during movement is most likely to flex at thin areas of the outer and ventral walls.

The mature wall contains a mottled layer sandwiched between two, more fibrillar layers. The internal wall layer has sublayers with fibrils in axial and radial orientations with respect to the pore.

During substomatal cavity formation, the middle lamella is stretched into an electron dense network and into strands and sheets.

After stomatal pore formation, the subsidiary cell walls close to the guard cell become strikingly thickened.

Preliminary observations indicate that two large vacuoles occupy the polar regions of open, but not closed, stomata.

Intact plasmodesmata occur in developing but not mature walls. Plastid structure, microtubule distribution, and other protoplasmic features are essentially similar to those described in the stomata of other genera.

Cuticle and pore development in the guard cells of Funaria were investigated with the electron microscope.

Pore cuticle formation is simultaneous with the creation of the pore itself. The morphology of the pore cuticle is unlike that of any cuticle described in the literature. It has many lamellae which are penetrated by electron dense fibrils.

Three different cuticular morphologies exist from the pore to the subsidiary cell walls. The cuticles on the pore and outer walls contain fibrils that sometimes reach to the surface.

The subsidiary cell cuticle lacks fibrils altogether. It is hypothesized that (1) cuticularization of the middle lamella contributes to ventral wall separation and (2) differences in extent of cuticular fibrils are related to greater water loss from stomata than from subsidiary cells (peristomatal transpiration).